The present invention relates to a new class of hydroxylic substrate bearing primary aromatic amine or diazo groups which can be used to bind nucleic acids, proteins, or other biological molecules functionally capable of reacting with diazotized primary amine groups, and various methods of preparation of such modified substrates.
The reactions of nucleic acids and proteins with cellulose papers modified with diazotized m-aminobenzyloxymethyl groups and some uses of such reactions have been described in a series of publications by Stark and collaborators in Cell 5, 301-310 (1975). Proc. Natl. Acad. Sci. U.S.A. 74, 5350-5354 (1977), Biochem Biophys. Res. Commun. 85, 1104-1112 (1978), Proc. Natl. Acad. Sci. U.S.A. 76, 3116-3120 (1979), and Proc. Natl. Acad. Sci. U.S.A. 76, 3683-3687 (1979). A paper modified with m-aminobenzyloxymethyl groups for the above purposes has been described by Rabbani, U.S. Pat. No. 4,139,346, Feb. 13, 1979.
The methods of Stark and collaborators and Rabbani rely upon a technology developed by Kursanov and Solodkov in J. Applied Chem. (U.S.S.R.) 16, 351-355 (1943) for the synthesis of m-nitrobenzyloxymethyl pyridinium chloride (NBPC), and its subsequent coupling to cotton fabric to secure an m-nitrobenzyloxymethyl derivative of cellulose. The m-nitrobenzyloxymethyl cellulose was reduced to the m-aminobenzyloxymethyl derivative, diazotized, and coupled to 2-napthol to afford a covalently dyed, red-colored fabric.
The advances of Stark and collaborators were (1) to demonstrate that the technology of Kursanov and Solodkov could be applied to couple nucleic acids and proteins to cellulose papers; and (2) specifically, to use such cellulose papers to immobilize patterns of nucleic acids and proteins in a form amenable to subsequent identification and analysis.
The Kursanov-Solodkov process, and its product, have several undesirable features. The NBPC reagent is expensive and difficult to obtain. The m-aminobenzyloxymethyl derivative of cellulose is unstable, and is best prepared just prior to use, a step which entails a tedious reduction and washing. Hence, it was an object of the present invention to devise a simple, economical synthesis of a stable paper substituted with aromatic primary amine or diazo groups.